Small Ubiquitin-related Modifier (SUMO) is a small protein posttranslational modification that occurs on lysine residues of proteins. It is reversibly conjugated to a wide range of cellular proteins through a multistep enzymatic cascade analogous to that of the ubiquitin conjugation pathway. The functional consequences of sumoylation are substrate specific and include changes in subcellular localization, protein-protein interactions, and transcriptional regulation. SUMO is involved in regulation of the cell cycle and the DNA damage response, thus linking SUMO to cancer progression. It is also involved in a variety of processes related to human disease: viral replication, Type 1 diabetes, inflammation, immunity and neurodegeneration. Understanding how sumoylation is regulated will help to further elucidate the pathophysiology of a wide range of human diseases. Presently, little is known about regulation of sumoylation in the global sense. The ubiquitylation cascade requires E1, E2, and E3 enzymes for substrate modification; moreover, substrate selection is determined by interplay between the E2 and E3 enzymes. Currently, sumoylation of many known substrates is only dependent on the E1 and E2 enzymes in vitro and it is commonly believed that E3 ligases merely enhance the degree of SUMOylation of target proteins rather than serve an essential role in target modification. However, emerging in vivo evidences suggest that the E3 ligases may play an important role in many sumo-dependent signaling pathways. Therefore, we hypothesize that a subset of SUMO-modified proteins may require the action of E3 ligases, thus conferring substrate specificity. We are also interested in interrogating the contribution of post-translational modification (PTM) crosstalk on SUMO substrate specificity. A small number of SUMO substrates have shown phosphorylation dependent sumoylation and a phosphorylation dependent sumoylation motif has been characterized. We hypothesize that both E3 ligases and phosphorylation by specific kinases contribute to SUMO substrate selection and regulation. We have established a global approach through the use of protein microarrays to identify substrates of the known SUMO E3 ligases. To elucidate the determinants of SUMO target selection, we propose to employ protein microarrays to identify E3- dependent SUMO substrates and to investigate crosstalk between sumoylation and phosphorylation, followed by in vivo characterization of selected targets and pathways.